Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment

PIP2-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations†

Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains

Модельные характеристики кардиореспираторной системы высококвалифицированных гребцов на байдарках и каноэ

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the "modeling by satisfaction of spatial restraints" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of σ values profoundly influences 3D modeling. We show that the σ values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the program's predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLER's objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance

Molecular dynamics simulations of a K+ channel blocker: Tc1 toxin from Tityus cambridgei.

Toxins that block voltage-gated potassium (Kv) channels provide a possible template for improved homology models of the Kv pore. In assessing the interactions of Kv channels and their toxins it is important to determine the dynamic flexibility of the toxins. Multiple 10 ns duration molecular dynamics simulations combined with essential dynamics analysis have been used to explore the flexibility of four different Kv channel-blocking toxins. Three toxins (Tc1, AgTx and ChTx) share a common fold. They also share a common pattern of conformational dynamics, as revealed by essential dynamics analysis of the simulation results. This suggests that some aspects of dynamic behaviour are conserved across a single protein fold class. In each of these three toxins, the residue exhibiting minimum flexibility corresponds to a conserved lysine residue that is suggested to interact with the filter domain of the channel. Thus, comparative simulations reveal functionally important conservation of molecular dynamics as well as protein fold across a family of related toxins

The intrinsic flexibility of the Kv voltage sensor and its implications for channel gating.

Analysis of the crystal structures of the intact voltage-sensitive potassium channel KvAP (from Aeropyrum pernix) and Kv1.2 (from rat brain), along with the isolated voltage sensor (VS) domain from KvAP, raises the question of the exact nature of the voltage-sensing conformational change that triggers activation of Kv and related voltage-gated channels. Molecular dynamics simulations of the isolated VS of KvAP in a detergent micelle environment at two different temperatures (300 K and 368 K) have been used to probe the intrinsic flexibility of this domain on a tens-of-nanoseconds timescale. The VS contains a positively charged (S4) helix which is packed against a more hydrophobic S3 helix. The simulations at elevated temperature reveal an intrinsic flexibility/conformational instability of the S3a region (i.e., the C-terminus of the S3 helix). It is also evident that the S4 helix undergoes hinge bending and swiveling about its central I130 residue. The conformational instability of the S3a region facilitates the motion of the N-terminal segment of S4 (i.e., S4a). These simulations thus support a gating model in which, in response to depolarization, an S3b-S4a "paddle" may move relative to the rest of the VS domain. The flexible S3a region may in turn act to help restore the paddle to its initial conformation upon repolarization